RECOMBINANT VP1 PROTEIN OF FMD VIRUS TYPE O/IRN/2010 AS AN IMMUNOGENIC PEPTIDE EXPRESSION SYSTEM ON GUINEA PIG
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Enterovirus 71 (EV71) is one of the viruses that cause hand, foot and mouth disease. Its viral capsid protein 1 (VP1), which contains many neutralization epitopes, is an ideal target for vaccine development. Recently, we reported the induction of a strong immune response in rabbits to a truncated VP1 fragment (Nt-VP1t) displayed on a recombinant Newcastle disease virus (NDV) capsid protein. Protective efficacy of this vaccine, however, can only be tested in mice, since all EV71 animal models thus far were developed in mouse systems. In this study, we evaluated the type of immune responses against the protein developed by adult BALB/c mice. Nt-VP1t protein induced high levels of VP1 IgG antibody production in mice. Purified VP1 antigen stimulated activation, proliferation and differentiation of splenocytes harvested from these mice. They also produced significant levels of IFN-γ, a Th1-related cytokine. Taken together, Nt-VP1t protein is a potent immunogen in adult mice and our findings provide the data needed for testing of its protective efficacy in mouse models of EV71 infections.
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To construct,express and purify the recombinant VP1 epitope vaccine for foot and mouth disease virus(FMDV)bovine O type and to investigate its immunological activity,the gene encoding the VP1 epitope of FMDV on B and T cells after screening the best epitope pattern on VP1 hexamer recombinant protein by computer analysis,was constructed by PCR and DNA cloning and then subcloned to plasmid pET28a(+).The target protein was expressed in E.coli with IPTG induction and purified by nicked affinity chromatography which was characterized by SDS-PAGE and ELISA.Cell neutralization test and the protection assay in neonate rats were used to detect the anti-FMDV antibody in sera of the immunized guinea pigs with VP1 epitope vaccine.In this way,the target gene was confirmed by sequence analysis and the expression yield of this protein was about 40% of the total bacterial protein.After purification by nicked affinity chromatography,the purity of this protein was about 90%.As demonstrated by ELISA,the anti-FMDV antibody could be detected in sera of the immunized guinea pigs.Neonate rats received sera of the immunized guinea pigs could be protected against challenge with living FMDV as revealed from the protection assay.From the above experimental results,it is concluded that the recombinant VP1 epitope vaccine of FMDV bovine O type can elicit the production of the protective neutralizing antibody against FMDV in guinea pigs which may serve as the candidate for the development of new type vaccine for FMDV infection.
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Objective To construct the DNA vaccine plasmids expressing the VP1 and VP4 genes of foot-and-mouth disease virus (FMDV)and study its immunogenicity. Methods The VP1 and VP4 genes of FMDV were amplified by RT-PCR, based on which the DNA vaccine plasmids for expression of VP1(pIRESneo-VP1), fusion expression of VP1 and VP4(pIRESneo-VP1VP4) and coexpression of VP1 and VP4(pIRESeno-VP1-VP4)were constructed and transfected to BHK-21 cells. The expressions of target proteins were identified by Western blot. Guinea pigs were immunized with recombinant plasmids pIRESneo-VP1, pIRESneo-VP1VP4, pIRESneo-VP1-VP4, pIRESneo-VP1 + pIRESneo-VP4, empty vector pIRESneo and inactivated FMDV vaccine respectively, determined for serum specific antibody and neutralizing antibody, then challenged with FMDV type O to evaluate the protection. Results PCR and restriction analysis proved that recombinant plasmids pIRESneo-VP1, pIRESneo-VP1VP4 and pIRESneo-VP1-VP4 were constructed correctly. All the DNA vaccines induced neutralizing antibody and showed a certain protective potency. Both the protective rates of guinea pigs in pIRESneo-VP1 and pIRESneo-VP1 + pIRESneo-VP4 groups were 28. 6%, and those in pIRESneo-VP1VP4 and pIRESneo-VP1-VP4 were 42. 9%, while those in inactivated FMDV and empty vector control groups were 100% and 0 respectively. Conclusion The FMDV containing both VP1 and VP4 showed high immunogenicity as compared with that containing VP1 only.
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Improving vaccine immunogenicity by targeting antigens to dendritic cells has recently emerged as a new design strategy in vaccine development. In this study, the VP1 gene of foot-and-mouth disease virus (FMDV) serotype A was fused with the gene encoding human immunodeficiency virus (HIV) membrane glycoprotein gp120 or C2-V3 domain of hepatitis C virus (HCV) envelope glycoprotein E2, both of which are DC-SIGN-binding glycoproteins. After codon optimization, the VP1 protein and the two recombinant VP1-gp120 and VP1-E2 fusion proteins were expressed in Sf9 insect cells using the insect cell-baculovirus expression system. Western blotting showed that the VP1 protein and two recombinant VP1-gp120 and VP1-E2 fusion proteins were correctly expressed in the Sf9 insect cells and had good reactogenicity. Guinea pigs were then immunized with the purified proteins, and the resulting humoral and cellular immune responses were analyzed. The VP1-gp120 and VP1-E2 fusion proteins induced significantly higher specific anti-FMDV antibody levels than the VP1 protein and stronger cell-mediated immune responses. This study provides a new perspective for the development of novel FMDV subunit vaccines.
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Hand-foot-and-mouth disease
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To develop a new type vaccine for Foot-and-Mouth Disease (FMD) prevention by using canine adenovirus as vector, the VP1 cDNA of Foot-and-Mouth Disease Virus (FMDV) type O strain China 99 was amplified by RT-PCR and cloned into pEGFP-C1 by replacing the GFP gene with the VP1 cDNA, resulting in an expression plasmid pVP1-C1. The expression cassette of VP1 composed of the CMV promoter, the VP1 gene and the SV40 early mRNA polyadenylation signal was recovered by Nsi I / Mlu I digestion of pVP1-C1 and cloned into the Canine adenovirus type-2 (CAV-2) genome in which E3 region was partly deleted by removing the Ssp I- Ssp I fragment. The recombinant virus (CAV-2-VP1) was obtained by transfecting the recombinant CAV-2-VP1 genome into MDCK cells with Lipofectamine™ 2000. Immunization trial in pigs with the recombinant virus, CAV-2-VP1, showed that CAV-2-VP1 could stimulate a specific immune response to both FMDV and the vector virus. Immune response to the VP1 and FMDV after VP1 expression was confirmed by ELISA, western blotting analysis and neutralization test. It was indicated that CAV-2 may serve as a vector for FMD vaccine development in pigs.
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